Topical application of spiperone or derivatives thereof for treatment of pathological conditions associated with immune responses

ABSTRACT

A method for the treatment of a cutaneous, ocular, or mucosal pathological condition which is associated with immune response in a human or other mammal, that includes topical application of an effective amount of spiperone or a spiperone derivative or its pharmaceutically acceptable salt, in a pharmaceutically-acceptable diluent or carrier for topical application.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part application of U.S. Ser. No.07/494,744, filed on Mar. 16, 1990, by Richard J. Sharpe, Kenneth A.Arndt, and Stephen J. Galli, which is a continuation-in-part applicationof U.S. Ser. No. 07/396,523, filed on Aug. 21, 1989 both abandoned.

This invention is in the area of the topical treatment of cutaneous,ocular, and mucosal hypersensitivity and hyperproliferative conditionsinduced by or associated with an immune response, that includes theapplication of an effective amount of spiperone or a spiperonederivative, or a pharmaceutically acceptable salt thereof, in apharmaceutically acceptable carrier.

The immune system specifically recognizes and selectively eliminatesforeign invaders, or other antigenic agents, by a process known as theimmune response. The immune response has three major characteristics: itresponds adaptively to foreign invaders, it exhibits strong specificity,and it displays a long-term memory of earlier contacts with specificforeign pathogens or antigens. The immune response involves theproduction of antibody and/or the destruction of antigenic cells bylymphocytes, which are highly specific for the antigen or hapten.

When directed against an infectious organism, the immune response canprovide great benefit to the host. As an example, an important componentof current public health practices is the use of vaccines to elicitimmune responses against infectious organisms that cause severe illnessand death. However, when directed against agents that are relativelyinnocuous, such as pollen, animal dander, and certain plant resins,damage to the host's tissues that is out of proportion to any threat tohealth posed by the antigenic agent that elicited the response can becaused by cells, antibodies, and mediators which represent the effectorcomponents of the immune response.

For example, cutaneous contact hypersensitivity responses are complexexpressions of a cellular immune response characterized byantigen-dependent changes in lymphocyte traffic, the recruitment ofcirculating leukocytes to the site of antigen challenge (leukocyteinfiltration) and alterations in vascular permeability and blood flowresulting in tissue swelling (edema). In humans and companion animals,cutaneous contact hypersensitivity responses can occur on exposure tocertain plant resins, such as those of poison ivy, and other commonlyencountered agents in the environment. In individuals sensitized to suchcommonly encountered agents, a severe contact reaction can result uponexposure, with significant associated morbidity. Severe or repeatedcontact hypersensitivity reactions can be followed by significantchronic changes, such as scarring of affected tissues, itchiness,swelling, scaling and oozing of tissue fluid through the skin surface.This pathology may predispose the patient to bacterial superinfection.In the eye, chronic immune responses can lead to diminished vision oractual blindness. In the lung, chronic immune responses, such as chronicallergic asthma, can result in serious chronic lung disease.

Cutaneous contact hypersensitivity and asthma are just two examples ofimmune responses that can be associated with significant morbidity.Others include atopic dermatitis, eczema, psoriasis, Sjogren's Syndrome,including keratoconjunctivitis sicca secondary to Sjogren's Syndrome,alopecia areata, allergic responses due to arthropod bite reactions,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, lichen planus, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, and drug eruptions. These conditions may result in any one ormore of the following symptoms or signs: itching, swelling, redness,blisters, crusting, ulceration, pain, scaling, cracking, hair loss,scarring, or oozing of fluid involving the skin, eye, or mucosalmembranes.

In atopic dermatitis, and eczema in general, immunologically mediatedleukocyte infiltration (particularly infiltration of mononuclear cells,lymphocytes, neutrophils, and eosinophils) into the skin importantlycontributes to the pathogenesis of these diseases. Chronic eczema alsois associated with significant hyperproliferation of the epidermis.Similarly, psoriasis, a common cutaneous disease associated with ahyperproliferating epidermis, also has a leukocyte infiltrationcomponent. Immunologically mediated leukocyte infiltration also occursat sites other than the skin, such as in the airways in asthma and inthe tear producing gland of the eye in keratoconjunctivitis sicca.

It is now believed that leukocytes and other cells found in the normaland abnormal skin, eye, or mucosal membranes secrete a variety ofcytokines. During immunological responses affecting these sites,cytokines are important in recruiting additional leukocytes into thesetissues, in promoting epithelial hyperproliferation, and in inducingother chronic changes such as scarring. For example, eosinophils, a typeof granulocyte found in many pathological immune responses includingatopic dermatitis and asthma, can product the cytokine TGF-α (Wong D. T.W., Weller P. F., Galli, S. J., Elovic A., Rand, T. H., Gallagher, G.T., Chiang, T., Chou, M. Y., Matossian, K., McBride, J., Todd, R. Humaneosinophils express transforming growth factor-alpha. J. Exp. med. 1990;172:673-81), which promotes epithelial hyperproliferation, and TGF-B(Wong, D. T. W., Elovic, A., Matossian, K., Nagura, N., McBride, J.,Chou, M. Y., Gordon, J. R., Rand, T. H., Galli, S. J., Weller, P. F.Eosinophils from patients with blood eosinophilia express transforminggrowth factor β1. Blood 1991; 78:2702-2707), which promotes fibrosis.

In addition to disorders that clearly represent pathologicalconsequences of immune responses, immune responses are thought tocontribute to many other pathological conditions, including Crohn'sdisease and ulcerative colitis of the gastrointestinal tract, psoriasis,alopecia areata and others. While the cause of most of these disordersis unclear, it is thought that exogenous agents yet to be defined orcomponents of the host's own tissues (in the case of autoimmunedisorders) may provoke an immune response that is responsible for theinfiltration of lymphocytes, monocytes, and granulocytes observed inthese conditions. It is also believed that the infiltrating cellssignificantly contribute to the tissue pathology associated with thesedisorders, through the production of cytokines as well as othermechanisms.

The need to control the wide variety of pathological responses withimmunological components which result in cutaneous, ocular, or mucosalhypersensitivity reactions, hyperproliferation, and scarring has led toa search for effective therapeutic agents that are both safe andeffective.

Because of the importance of leukocytes and their products in thedevelopment of pathology associated with immune responses, manyapproaches to treating these conditions are focused on inhibiting theimmune responses and leukocyte infiltration contributing to thesedisorders. Several substances are known to be able to inhibit the immuneresponses contributing to cutaneous leukocyte responses orhyperproliferative responses. Corticosteroids, when administeredsystemically, are effective in this regard but are associated withsignificant and potentially dangerous side effects. Topically appliedcorticosteroids have some efficacy in treating these conditions, but areonly partially effective in many instances and have their ownsignificant side effects, including atrophy of tissue, formation oftelangiectasia, blanching, and a myriad of systemic effects ifsignificantly absorbed. Other agents with partial utility for treatingsome of the above conditions include psoralen plus ultraviolet A (PUVA),cyclosporin A, or azathioprine, but the risk-to-benefit ratios for theseagents is unfavorable for most of the conditions described above.

As a result, there is a significant and very longstanding need toidentify new agents with favorable benefit to risk ratios that can beapplied topically to prevent or suppress (i.e. "treat") immune responsescontributing to cutaneous, ocular, or mucosal hypersensitivityreactions, hyperproliferation, or scarring. Optimally, such agentsshould be effective when applied locally, and systemic absorption shouldnot result in blood levels high enough to cause significant systemictoxicity or other adverse side effects. Not only does localadministration place the agent in closest contact with the site needingtreatment, but it also diminishes the possibility that such treatmentwill suppress beneficial immune responses which may occur at other, moredistant, sites.

In contrast to the immune response, an inflammatory response is apathologic condition that can occur in response to immunologicallynon-specific injury, either from physical (such as trauma), chemical, orbiologic agents. An inflammatory response is characterized by increasedblood flow and redness in the inflamed area, increased capillarypermeability and edema, and recruitment of immunologically non-specificwhite blood cells, especially neutrophils, that remove injuriousmaterial and promote repair. Unlike immune responses, inflammatoryresponses do not respond adaptively to the inciting stimulus, do notshow specificity and do not exhibit long term memory. Cellular productsof lymphocytes may contribute to or induce an inflammatory response.However, because of the differences in mechanisms, a compound canfunction as an anti-inflammatory agent without having immunosuppressiveproperties. Phenylbutazone, indomethacin, aspirin, ibuprofen, andacetaminophen are examples of anti-inflammatory compounds which have nosignificant immunosuppressive activity, as demonstrated by their lack ofa significant effect on immunologically mediated responses, such ascontact hypersensitivity. U.S. Pat. No. 3,996,363 to Wade discloses thatcertain naphthalimide derivatives of spiperone have anti-inflammatoryactivity.

Spiperone(8-[3-(p-fluorobenzoyl}propyl]-1-propyl]-1-phenyl-1,3,8-triazaspiro-[4.5]decan-4-one)is a neuroleptic agent with central nervous system (CNS) dopamine andserotonin (5-HT) receptor antagonist properties. Some analogues ofspiperone are useful as experimental reagents in dopamine and serotoninreceptor studies. For example, the high affinity of an immobilizedspiperone derivative,3-(2-aminoethyl)-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-onetrihydrochloride, for dopamine receptors has made it possible to isolatethese receptors in pure form. Radiopharmaceuticals based on spiperoneand its analogues have been shown to be useful in assessing dopaminereceptor function based on positron emission tomography (PET) in animalsand man. Spiperone has also been shown to bind to human and mouselymphocytes, although the mechanism responsible for such binding isuncertain.

U.S. Pat. No. 4,874,766 assigned to Janssen Pharmaceutica N.V. disclosesa method for promoting wound-healing by topical administration of aserotonin-antagonist compound, including spiperone and its derivatives.Wound healing is a reparative process by which several types of residentcells, such as epithelial cells, fibroblasts and vascular endothelialcells, and certain circulating cells, including neutrophils, lymphocytesand macrophages, act in concert to restore to a more healthy conditiontissues that have sustained various forms of mechanical or other injury.Although lymphocytes and macrophages participate in both wound healingand in immune responses, the specific roles of these cells in the twotypes of processes may be distinct. In fact, treatment of wounds withimmunosuppressive agents, such as corticosteroids and cyclosporin A, hasbeen known to cause impairment of the healing process (Arch Suro May1990, 125(5), 636-40; Ann Ophthamol. April 1985; 17(4), 238, and J.Suro. Res. June 1983; 34(6), 572-5).

It is an object of the present invention to present a method for thetopical treatment of cutaneous, mucosal and ocular pathology associatedwith immune responses.

It is yet another object of the present invention to present a methodfor the topical treatment of cutaneous, mucosal, or ocularhypersensitivity and epithelial hyperproliferation.

It is yet another object of the invention to present a method for thetopical treatment of cutaneous, mucosal or ocular scarring.

SUMMARY OF THE INVENTION

A method for the treatment of a cutaneous, ocular, or mucosal conditionin a human or other mammal resulting from pathology associated with animmune response, that includes topical application of an effectiveamount of spiperone or a spiperone derivative or its pharmaceuticallyacceptable salt, in a pharmaceutically-acceptable diluent or carrier fortopical application.

It has been discovered that the parent spiperone exhibits a strongimmunosuppressive activity when applied topically. The parent spiperoneis used herein as the model of an active topical immunosuppressant.Spiperone derivatives are measured against this model, and areconsidered to be immunosuppressants if they suppress the leukocyteinfiltration and/or the ear swelling associated with an experimentalcontact hypersensitivity response by at least 40% at 24 hours afterspecific antigen challenge.

In the preferred method of administration, the active compounds areadministered topically in a suitable carrier to effectivelyimmunosuppress the patient at the site of application. Because theapplication is topical, i.e., local, immunosuppression is achievedwithout producing systemic effects, most notably, the significantneuroleptic effect that is associated with the systemic administrationof spiperone.

Spiperone and its active derivatives are useful as topical agents intreating contact dermatitis, atopic dermatitis, eczematous dermatitis,psoriasis, Sjogren's Syndrome, including keratoconjunctivitis siccasecondary to Sjogren's Syndrome, alopecia areata, allergic responses dueto arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, and drug eruptions. The novel method may also be useful inreducing the infiltration of skin by malignant leukocytes in diseasessuch as mycosis fungoides. These compounds can also be used to treat anaqueous-deficient dry eye state (such as immune mediatedkeratoconjunctivitis) in a patient suffering therefrom, by administeringthe compound topically to the eye.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Effects of systemic spiperone (30 or 150 mg/kg, subcutaneously)on the tissue swelling associated with oxazolone-induced cutaneouscontact hypersensitivity reactions. Spiperone or vehicle alone (0) wasadministered to C57BL/6J mice 1 hour after challenge for contacthypersensitivity. The change in ear thickness (post-challenge valueminus baseline prechallenge value) was measured 24 hours after oxazolonechallenge. The data are presented as the mean ±SEM. The reduction in earswelling observed with either 30 or 150 mg/kg spiperone was significantwhen compared to the reactions observed in the control animals(**=p<0.01).

FIG. 2 Effects of systemic treatment with 30 or 150 mg/kg spiperone,subcutaneously, on leukocyte infiltration associated with 24-hourcontact hypersensitivity reactions. These data (mean ±SEM) are derivedfrom the same mice whose ear thickness values are shown in FIG. 1. Thereduction in leukocyte infiltration observed in animals treated with 30or 150 mg/kg spiperone was significant when compared to the reactionsobserved in animals treated with vehicle alone (* or **=p<0.05 or 0.01,respectively).

FIG. 3 Comparative effects of systemic vehicle (1), haloperidol (2),trazadone (3), mianserin (4) or spiperone (5) (all agents at 40 mg/kg,subcutaneously) on the tissue swelling associated with oxazolone-inducedcutaneous contact hypersensitivity reactions. Spiperone, the otheragents, or vehicle alone were administered to BALB/c mice 1 hour afterchallenge for contact hypersensitivity. The change in ear thickness(post-challenge value minus baseline pre-challenge value) was measured24 hours after oxazolone challenge. The data are presented as the mean±SEM. The reduction in ear swelling observed with spiperone wassignificant when compared to the reactions observed in the control,vehicle treated animals (**=p<0.01), whereas haloperidol, trazadone andmianserin did not significantly suppress the tissue swelling associatedwith contact hypersensitivity.

FIG. 4 Comparative effects of systemic treatment with vehicle (1) orhaloperidol (2), trazadone (3), mianserin (4) or spiperone (5) (allagents at 40 mg/kg), administered subcutaneously, on leukocyteinfiltration associated with 24-hour contact hypersensitivity reactions.These data (mean ±SEM) are derived from the same mice whose earthickness values are shown in FIG. 3. The reduction in leukocyteinfiltration observed in animals treated with spiperone was significantwhen compared to the reactions observed in animals treated with vehiclealone (*p<0.05), while haloperidol, trazadone and mianserin did notsignificantly suppress the leukocyte infiltration associated withcontact hypersensitivity.

FIG. 5 Effect of spiperone applied topically during the period ofsensitization on the tissue swelling associated with oxazolone-inducedcontact hypersensitivity reactions. Oxazolone was applied to theabdomens of BALB/c mice on day 0. The change in ear thickness wasdetermined 24 hours after challenge with oxazolone on day 6. Treatmentwith spiperone (50 μl of 0.08% spiperone in propylene glycol) applied tothe abdomens on days -2, -1, 0, 1 and 2 significantly diminished contacthypersensitivity reactions in the right ears of the treated animals(**p<0.01 when compared to the right ears in the control mice treatedwith vehicle).

FIG. 6 Effect of spiperone applied topically during the period ofsensitization on the leukocyte infiltration associated withoxazolone-induced contact hypersensitivity reactions. These data (mean±SEM) are from the same mice whose ear thickness measurements arepresented in FIG. 5. Topical treatment with spiperone significantlydiminished the reactions when compared to those in vehicle-treated mice(**p<0.01).

FIGS. 7a,b,c Effect of topically administered spiperone on tissueswelling associated with oxazolone-induced contact hypersensitivityreactions. Oxazolone was applied to both ears of all mice and the changein ear thickness was measured at a specified interval thereafter. a. Onehour after oxazolone challenge, 4.0% spiperone in ethanol:propyleneglycol:olive oil was applied to both surfaces of the right ears of somemice, whereas vehicle alone was applied to both surfaces of the rightears of the control (0% spiperone) mice. The ears were measured 24 hoursafter oxazolone challenge. Local treatment with 4% spiperone suppressedswelling in the treated ear (**=p<0.01 vs either contralateral oxazolonetreated ears or ears of vehicle treated group) and also diminished theswelling in the contralateral ears (**=p<0.01 vs left ears of vehicletreated group), although to a lesser extent than in the treated ears. b.Two hours before oxazolone challenge, 0.13% spiperone in Vehicle-N wasapplied to both surfaces of the right ears of some mice, whereas vehiclealone was applied to both surfaces of the ears of the control (0%spiperone) animals. The ears were measured 24 hours after oxazolonechallenge. Local treatment of the right ear with spiperone significantlysuppressed tissue swelling in the treated ear (**p<0.01 vs contralateraloxazolone treated ears or vs right ears of vehicle treated group).However, treatment of the right ear with 0.13% spiperone had nosignificant effect on the magnitude of swelling in the contralateraloxazolone treated ear. c. Twenty-two hours after oxazolone challenge,0.13% spiperone in Vehicle-N was applied to both surfaces of the rightears of some mice, whereas vehicle alone was applied to both surfaces ofthe ears of control (0% spiperone) mice. The change in ear thickness wasdetermined 24 hours after treatment with spiperone, i.e. at 46 hoursafter challenge with oxazolone. Treatment with spiperone significantlydiminished contact hypersensitivity reactions in the right ears of thetreated animals (*=p<0.01 when compared to the right ears {in thecontrol mice, and p<0.05 when compared to the contralateral ears of thesame mice). The reactions in the left ears of the mice treated on theright ears with spiperone were not reduced when compared to reactions inthe left ears of the vehicle-treated mice.

FIGS. 8a,b,c Effect of topical treatment with spiperone on leukocyteinfiltration associated with oxazolone-induced contact hypersensitivityreactions. These date (mean ±SEM) are from the same mice whose earthickness measurements are presented in FIGS. 7a,b,c. Biopsies wereperformed 24 hours (a, b) or 46 hours (c) after application ofoxazolone. Topical treatment with spiperone significantly diminished thereactions when compared to those in vehicle-treated mice (II=p<0.01). InFIG. 8a, the slight effect of treatment of the right ears with spiperoneon reactions expressed in the left ear of the same mice was notsignificant (p>0.05).

FIG. 9 Effect of topically administered spiperone on tissue swellingassociated with DNFB-induced contact hypersensitivity reactions. DNFBwas applied to both ears of C57L/6J mice. One hour later, 0.5% spiperonewas applied to both surfaces of the right ears of some mice, whereasvehicle alone was applied to both surfaces of the right ears of thecontrol (0% spiperone) mice. The change in ear thickness was determined24 hours after challenge with DNFB. Treatment with spiperonesignificantly diminished contact hypersensitivity reactions in the rightears of the treated animals (**p<0.01 when compared to the right ears incontrol mice, and p<0.05 when compared to the contralateral ears of thesame mice). The reactions in the left ears of the mice treated on theright ears with spiperone were also reduced slightly when compared toreactions in the left ears of the vehicle-treated mice (*p<0.05).

FIG. 10 Effect of topical treatment with spiperone on leukocyteinfiltration associated with DNFB-induced contact hypersensitivityreactions. These data (mean ±SEM) are from the same mice whose earthickness measurements are presented in FIG. 9. Topical treatment withspiperone significantly diminished the reactions when compared to thosein vehicle-treated mice (**p<0.01). The slight effect of treatment ofthe right ears with spiperone on reactions expressed in the left ears ofthe same mice was not significant (p>0.05).

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As used herein, the term "spiperone" refers to the compound(8-[3-(p-fluorobenzoyl)propyl]-1-propyl]-1-phenyl1,3,8-triazaspiro-[4.5]decan-4-one).

As used herein, the term "spiperone derivative" refers to a molecule (1)that contains the spiperone nucleus: ##STR1## wherein R₁ =H, CH₃ --, C₆H₅ --, cyclohexyl, 4--(OCH₃)C₆ H₄ --, 3--(CH₃)C₆ H₄ --, 4--(CH₃)C₆ H₄--, 4-X-C₆ H₄ --, (CH₃)₂ CH--, CH₃ (CH₂)₃ --, (CH₃)₂ CHCH₂ --, CH₃ CH₂CH(CH₃)--, (CH₃)₃ C--; or Y--CH₂ (CH₂)_(m) --.

R₂ =H or CH₃ ;

R₃ =H, CH₃, CH₃ CH₂ --, CH₃ CH₂ CH₂ --, (CH₃)₂ CH--, CN(CH₂)₂ --, or CH₃(CH₂)_(s) --;

R₄ =H, C₆ H₅ CH(CH₂ CH₃)CH₂ --, C₆ H₅ CH(CH₃)(CH₂)₂ --, C₆ H₅ CH₂CH(CH₃)CH₂ --, C₆ H₅ CH₂ CH₂ CH(CH₃)--, C₆ H₅ CH(CH₃)(CH₂)₃ --,4--CH₃ C₆H₄ CH(CH₃)(CH₂)₃ --, 4--(CH₃ O)C₆ H₄ CH(CH₃)(CH₂)₃,4--X--C₆ H₄CH(CH₃)CH₂ --, 4--X--C₆ H₄ CH(CH₂ CH₃)CH₂ --,4--X--C₆ H₄ CH(CH₃)(CH₂)₂--, 4--X--C₆ H₄ --CH(CH₃)(CH₂)₃ --,C₆ H₅ CH(OCH₃)(CH₂)₂ --, ##STR2## C₆H₅ CO(CH₂)₃ --,C₆ H₅ CO(CH₂)₄ --, 4--(CH₃)C₆ H₄ CO(CH₂)₃ --, 4--(CH₃O)C₆ H₄ CO(CH₂)₃ --, 4--X--C₆ H₄ CO(CH₂)₃ --, 4--X--C₆ H₄ CO(CH₂)₃ --,2--thienyl--CO(CH₂)₃ --, ##STR3##

4--XC₆ H₄ C(CH₃)CH(CH₂)₂ --, where the configuration about the doublebond is cis or trans,

4-XC₆ H₄ C(CH₃)CHCH₂ --, where the configuration about the double bondis cis or trans,

4--X--C₆ H₄ COCH=CHCH₂ --, or

Y--CH₂ (CH₂)_(s--).

wherein n =3 or 4; m is between 1 to 4; s is between 1 to 6; X =H or isa heteroatom or a substituted heteroatom such as F, Cl, Br, I, OCH₃,SO₃, or NHz; Y=H or a heteroatom such as F, Cl, Br, I, SO₃, PO₄ ⁼, OH,SH, SCH₃, CH₃ SO₂, NH₂, -CO₂ ; and each of Ar and Ar₁ is, independently,H, C₆ H₅ --, 4--(CH₃)C₆ H₄ --, 4--(CH₃ O)C₆ H₄ --, 4--X--C₆ H₄ --,3--(CH₃)C₆ H₄ --, 2-thienyl, or 4--X--C₆ H₄ CH₂ --;

and (2) exhibits an immunosuppressive effect when provided topically, asmeasured using the assay set out in Example I, i.e., they suppress theleukocyte infiltrate and/or the ear swelling associated with anexperimental contact hypersensitivity response by at least 40% at 24hours after specific antigen challenge, or as evaluated in vivo inhumans by the agent's ability to inhibit contact hypersensitivityresponses to patch test allergens in patients hypersensitive to a givenallergen, using procedures generally accepted by those of skill in theart.

As illustrated in FIGS. 3 and 4, the chemically unrelated serotoninreceptor antagonists, trazadone and mianserin, and the dopamine receptorantagonist, haloperidol, are not effective in suppressing contacthypersensitivity. On this basis, it is clear that the mechanism ofaction of spiperone and spiperone derivatives in suppressing the immuneresponse is independent of their serotonin or dopamine receptor blockingproperties.

I. Structure and Synthesis of Spiperone Derivatives

The parent spiperone is8-[3-(p-fluorobenzoyl)propyl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one,which has the structure illustrated below. ##STR4##

As demonstrated in Example 1, the parent spiperone has significantimmunosuppressive activity when applied topically. The potential utilityof any one of the above-described spiperone derivatives to act as animmunosuppressant can be conveniently determined by synthesizing thecompound and testing it in the biological assay described in Example 1.

Those derivatives of spiperone which are particularly useful in themethod of the invention are those which have decreased affinities fordopamine and/or serotonin receptors, but which obtain immune suppressiveproperties.

Methods of synthesis of spiperone derivatives are disclosed in, or canbe easily adapted from syntheses disclosed in, U.S. Pat. Nos. 3,155,669;No. 3,155,670; No. 3,161,644; and No. 3,238,216; all of which are herebyincorporated by reference.

III. Therapeutic Compositions

Mammals, and specifically humans, suffering from pathogenic cutaneous,ocular, or mucosal immune responses can be treated by topicaladministration to the patient of an effective amount of the spiperonederivative or its salt in the presence of a pharmaceutically acceptablecarrier or diluent.

The active compound is administered topically in an effective dosagerange to cause immunosuppression of the target pathogenic immuneresponse. The active compound is included in the pharmaceuticallyacceptable carrier or diluent in an amount sufficient to deliver to apatient a therapeutic amount of the spiperone derivative in vivo in theabsence of serious toxic effects. In general, local immunosuppressioncan be achieved by topically administering lower doses of spiperonederivatives than would be required if the agents were administeredsystemically.

A typical daily dose of active compound is between 0.1 milligrams and120 grams. The active compounds can be applied in any effectiveconcentration, usually varying between 0.001% and 50% (all percentagesare by weight).

Spiperone or its derivative is administered for a sufficient time periodto alleviate the undesired symptoms and the clinical signs associatedwith the condition being treated. The concentration of active compoundin the drug composition will depend on absorption, inactivation, andother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition. Theactive ingredient may be administered at once, or may be divided into anumber of smaller doses to be administered at varying intervals of time.

The spiperone derivative can be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antibiotics, antifungals, anti-inflammatories,antivirals, or other immunosuppressive agents.

Solutions or suspensions for topical application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Suitable vehicles or carriers for topical application are known, andinclude lotions, suspensions, ointments, creams, gels, tinctures,sprays, powders, pastes, slow-release transdermal patches, aerosols forasthma, suppositories for application to rectal, vaginal, nasal or oralmucosa, moothwashes, or swish and split preparations.

Thickening agents, emollients, and stabilizers can be used to preparetopical compositions. Examples of thickening agents include petrolatum,beeswax, xanthan gum, or polyethylene glycol, humectants such assorbitol, emollients such as mineral oil, lanolin and its derivatives,or squalene. A number of solutions and ointments are commerciallyavailable, especially for ophthalmic and dermatologic applications.

Natural or artificial flavorings or sweeteners can be added to enhancethe taste of topical preparations applied for local effect to mucosalsurfaces. Inert dyes or colors can be added, particularly in the case ofpreparations designed for application to oral mucosal surfaces.

Spiperone or its derivative can be applied in a time release formulationvia patches or by slow release polymers. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. The materials can also beobtained commercially from Alza Corporation and Nova Pharmaceuticals,Inc.

Spiperone or its derivatives can be provided in the form ofpharmaceutically-acceptable salts. As used herein, the term"pharmaceutically-acceptable salts or complexes" refers to salts orcomplexes that retain the desired biological activity of the parentcompound and exhibit minimal, if any, undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid, and the like), and salts formed withorganic acids such as acetic acid, oxalic acid, tartaric acid, succinicacid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acids,naphthalenedisulfonic acids, and polygalacturonic acid; (b) baseaddition salts formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium, and the like, or with an organic cation formed fromN,N-dibenzylethylene-diamine or ethylenediamine; or (c) combinations of(a) and (b); e.g., a zinc tannate salt or the like.

The spiperone derivatives can be modified in order to enhance theirusefulness as pharmaceutical compositions. For example, it is well knowin the art that various modifications of the active molecule, such asalteration of charge, can affect water and lipid solubility and thusalter the potential for percutaneous absorption. The vehicle, orcarrier, can also be modified to enhance cutaneous absorption, enhancethe reservoir effect, and minimize potential irritancy orneuropharmacological effects of the composition. See, in general, Arndt,K. A., P. V. Mendenhall, "The Pharmacology of Topical Therapy",Dermatology in General Medicine, 1987; T. B. Fitzpatrick, A. Z. Eisen,K. Wolff, I. M. Freedberg and K. F. Austen, eds., 3d ed., McGraw Hill,Inc., N.Y., pp. 2532-2540.

IV. Immunosuppressant Activity of Spiperone Derivatives

Spiperone and spiperone derivatives are capable of suppressing theimmune response in humans and other mammals on topical application. Assuch, the compounds, or therapeutic compositions thereof, are useful forthe treatment of a myriad of immunological disorders. Pathogenic immuneresponses that can be treated by topical application of spiperone orspiperone derivatives include contact dermatitis, atopic dermatitis,eczematous dermatitis, drug eruptions, lichen planus, psoriasis,alopecia areata, Sjogren's Syndrome, including keratoconjunctivitissicca secondary to Sjogren's Syndrome, cutaneous lupus erythematosus,scleroderma, allergic reactions secondary to arthropod bite reactions,aphthous ulcers, conjunctivitis, keratoconjunctivitis, iritis, asthmaand allergic asthma, vaginitis, Crohn's disease, ulcerative colitis andproctitis. These compounds can also be useful in reducing theinfiltration of skin by malignant leukocytes in diseases such as mycosisfungoides.

Spiperone and its derivatives can also be used to increase tearproduction in a patient suffering from deficient tears in the eye due toan autoimmune dysfunction of the lacrimal glands, such as immunemediated keratoconjunctivitis (KCS, or dry eye). Canine KCS is a common,chronic progressive, and potentially blinding disease. A continuum ofcorneal and conjunctival lesions ensues from the dry eye state.Spiperone or its active derivatives can be provided as an ophthalmicdrop or ophthalmic ointment to humans or other mammals, including dogsand cats, in an effective amount in a suitable vehicle. This topicalophthalmic treatment can also serve to correct corneal and conjunctivaldisorders exacerbated by tear deficiency and KCS, such as cornealscarring, corneal ulceration, filamentary keratitis, mucopurulentdischarge, and vascularization of the cornea. Spiperone and itsderivatives can also be used to decrease immune responses whichcontribute to granulation and neovascularation in the cornea.

The ability of spiperone(8-[3-{p-fluorobenzoyl}propyl]-1-phenyl-1,3,8-triazaspiro-[4.5]decan-4-one)to influence the tissue swelling and leukocyte infiltration associatedwith contact hypersensitivity reactions in mice was evaluated asdescribed in detail in Example 1. The parent spiperone compound was usedfor the procedure in Example 1 as a model of an activeimmunosuppressant. Spiperone derivatives can be measured against thismodel, and are considered active if they suppress the leukocyteinfiltrate and/or the swelling response by at least 40% 24 hours afterspecific antigen challenge.

In the procedure of Example 1, contact hypersensitivity reactions wereelicited by applying the haptens oxazolone or dinitrofluorobenzenetopically to one or both ears five to eight days after epicutaneoussensitization. When spiperone was given subcutaneously at a dose of 150mg/kg, 1 hour after challenge with oxazolone, cutaneous contacthypersensitivity to this hapten was almost totally abrogated.

This result indicates that spiperone can virtually eliminate theexpression of contact sensitivity in subjects that have previously beensensitized to develop an immunological response by exposure to the agentoxazolone. This test mimicks the common clinical situation in whichpatients who have developed sensitivity to an agent seek relief from theexpression of contact sensitivity in response to subsequent encounterswith that agent.

A dose of 40 or 30 mg/kg of spiperone subcutaneously also significantlysuppressed the reactions but to a lesser degree than the higher dose.When applied topically, preparations of spiperone significantlysuppressed both the tissue swelling and the leukocyte infiltrationassociated with the elicitation phase of contact hypersensitivity toeither oxazolone or dinitrofluorobenzene. Topical treatment withspiperone also suppressed the sensitization phase of contactsensitivity. However, mice treated topically with spiperone, unlikethose treated systemically, exhibited no drowsiness or other evidence ofcentral nervous system effects.

Spiperone expresses both serotonin and dopamine receptor antagonistactivity. However, unlike spiperone, it was discovered that thechemically unrelated serotonin antagonists, trazadone and mianserin, andthe dopamine receptor antagonist, haloperidol, were not effective insuppressing contact hypersensitivity. On the basis of this, it is clearthat the mechanism of action of spiperone on the immune response isindependent of its serotonin or dopamine receptor blocking properties.

EXAMPLE 1 Inhibition of Induced Contact Hypersensitivity.

Six-to-8-week-old female C57BL/6J or BALB/c mice were obtained from theJackson Laboratory, Bar Harbor, Maine or from Charles RiverLaboratories, Kingston Facility, Stoneridge, N.Y., respectively.

Spiperone, mianserin, trazadone, haloperidol and oxazolone werepurchased from the Sigma Chemical Co. (St. Louis, Mo.).

Oxazolone-Induced Contact Hypersensitivity

Sensitization and challenge for contact hypersensitivity were performedas follows. The abdomens of the mice were shaved with electric clippers,50 μl of a 4% (w/w) solution of oxazolone in 4:1 (v:v) acetone:olive oilwere applied to the shaved abdomen, and 5 μl of the same solution wereapplied to each hind footpad. Five to eight days later, the mice werechallenged for contact hypersensitivity by applying 10 μl of a 0.5%(w:w) solution of oxazolone in 4:1 (v:v) acetone:olive oil to both theinner and outer surface of the right ear of each mouse (in the case ofmice treated systemically with spiperone) or to both ears (in the caseof mice treated topically with spiperone).

Dinitrofluorobenzene-Induced Contact Hypersensitivity

Mice were treated in an identical manner as above, except that 0.2%(v:v) 1-fluoro-2,4-dinitrobenzene (DNFB) in acetone was used for bothsensitization and elicitation of the contact hypersensitivity response.

Systemic Spiperone Treatment

One hour after the application of oxazolone for elicitation of contacthypersensitivity, mice were treated subcutaneously with spiperone (150or 30 mg/kg body weight) in 0.1 ml of carrier (Cremophor EL, BASF,Parsippany, N.J.), or with 0.1 ml of carrier alone. In a separateexperiment, mice were treated in a similar fashion with 40 mg/kg bodyweight of trazadone, mianserin, haloperidol, or spiperone in 0.1 mlolive oil or with olive oil alone.

Topical Spiperone Treatment

To test whether spiperone affected the sensitization phase of contacthypersensitivity, 50 μl of 0.08% spiperone in propylene glycol wasapplied to the shaved abdomens of the mice on days -2, -1, 0, 1 and 2,with the day of oxazolone sensitization being designated day 0. To testthe effects of spiperone on the expression of contact hypersensitivityin mice already sensitized to oxazolone; mice were treated withspiperone topically at two hours before or one or twenty-two hours afterchallenge for contact hypersensitivity, by applying 10 μl of a solutionof spiperone in vehicle to both sides of the right ear. In the case ofoxazolone-sensitized mice treated one hour after challenge, a 4% (w/w)spiperone suspension in 4:1:5 absolute ethanol:propylene glycol:oliveoil was used, while 0.13% (w/w) spiperone solution in Vehicle-N(Neutrogena Corp., Los Angeles, CA) was used at the other time points.In the case of the DNFB-sensitized mice, 0.5% (w/w) spiperone inabsolute ethanol was used.

Evaluation of Ear Swelling Response

Immediately before and 24 or 46 hours after application of oxazolone orDNFB, ear thicknesses were determined with an engineer's micrometer. Theincrement (delta) in ear thickness (ear swelling) was calculated as the24- or 46-hour value minus the baseline (prechallenge) value andexpressed in units of 10⁻² mm. Mice were killed by cervical dislocationafter the measurement of 24 or 46-hour ear thickness was obtained, andthe ears were processed for histologic examination.

Quantification of Leukocyte Infiltration

In most experiments, both ears of each mouse were fixed in 4.0% bufferedformalin and then processed routinely and embedded in paraffin forpreparation of 6-7 μm-thick hematoxylin and eosin-stained sections. Insome experiments (FIGS. 2 and 10), ears were fixed and processed into 1μm thick, Epon-embedded, Giemsa-stained sections. All of the sectionswere coded and examined with an ocular grid at 400× under lightmicroscopy by an observer unaware of the identity of the individualslides. The number of leukocytes/mm² of dermis was calculated bycounting all of the leukocyte cells in an area of at least 0.14 mm² ofdermis.

Statistical Analysis

Differences between groups were assessed by the 2-tailed Student's ttest (paired for comparisons of left and right ears in the same mice,unpaired for comparisons between different groups of mice).

Results Effects of Systemic Treatment with Spiperone on Expression ofContact Hypersensitivity

The subcutaneous administration of spiperone at a dose of 150 mg/kg, 1hour after challenge for contact hypersensitivity to oxazolone, markedlydiminished (by 80%) the tissue swelling which developed in associationwith the contact hypersensitivity response (FIG. 1). FIG. 2 shows thatthe leukocyte infiltration associated with the response in mice treatedwith 150 mg/kg spiperone was also diminished by approximately the sameamount (81% reduction compared to responses in mice not treated with thedrug). However, at this dose, spiperone also produced other remarkablesystemic effects. The mice rapidly became lethargic after administrationof the drug, and, by 23 hours after spiperone injection, the miceexhibited profound depression of central nervous system function. Theyappeared to be in a deep sleep, neither ate nor drank, and respondedweakly or not at all to touch. They did, however, exhibit responsivenessto pinch.

Some mice were treated with spiperone at 30 mg/kg subcutaneously (FIGS.1 and 2). At this dose, spiperone diminished the tissue swellingassociated with contact hypersensitivity to oxazolone to almost the sameextent as did the higher dose (68% reduction with 30 mg/kg versu 80%reduction with 150 mg/kg) but reduced the leukocyte infiltrationassociated with the reaction by only 37% (FIG. 2). However, the centralnervous system effects of spiperone at 30 mg/kg were substantially lesspronounced that those observed at the higher dose. Thus, the micetreated with spiperone at 30 mg/kg were less sleepy than those treatedwith 150 mg/kg. However, the mice treated with 30 mg/kg appearedsomewhat lethargic and were less interested in food and water than werecontrol mice treated with carrier alone.

Systemic Spiperone Versus Other Serotonin or Dopamine ReceptorAntagonists

In these experiments, systemic spiperone was compared to the serotoninreceptor antagonists, trazadone or mianserin, and to the dopaminereceptor antagonist, haloperidol, for their ability to inhibit cutaneouscontact hypersensitivity. At a dose of 40 mg/kg, only systemic spiperonesignificantly reduced cutaneous contact hypersensitivity (FIG. 3, 4).The degree of lethargy in mice treated with 40 mg/kg of spiperone,trazadone, mianserin or haloperidol systemically (FIG. 1 and 2),appeared to be about the same as that in the mice treated with 30 mg/kgof spiperone systemically.

Effects of Spiperone on the Sensitization Phase of ContactHypersensitivity

For these experiments, mice were treated topically with spiperone inVehicle-N or Vehicle-N alone, applied to the abdomen beginning two daysprior to sensitization and continuing for a total of 5 days (FIGS. 5 and6). Mice treated with spiperone exhibited 64% less tissue swelling and70% less leukocyte infiltration at sites of hapten challenge than didshow that treatment with topical spiperone can effectively inhibit thesensitization phase of cutaneous contact hypersensitivity.

Effects of Topical Spiperone on Expression of Contact Hypersensitivity

For these experiments, both ears of each mouse were challenged forelicitation of contact hypersensitivity by the application of oxazoloneor DNFB (as appropriate) to both surfaces of both ears. Two hoursbefore, one hour after or twenty-two hours after application of hapten,the right ears of some mice were treated with spiperone in vehicle,applied epicutaneously (topically to the skin) to both surfaces of theears. The right ears of control mice were similarly treated, but withvehicle alone. Topical administration of a 4.0% suspension of spiperonein absolute ethanol, propylene glycol, and olive oil one hour afterhapten challenge resulted in a marked diminution of the tissue swellingassociated with contact hypersensitivity reactions elicited in the right(spiperone-treated) ear and had a smaller, but nonetheless significant,effect on the swelling associated with the contact hypersensitivityreaction elicited on the contralateral (untreated) ear (FIG. 7a). Thus,reactions in the untreated right ears were 90% smaller than reactions inthe right ears of vehicle-treated mice, whereas reactions in the leftears of mice treated on the right ears with spiperone were reduced by60% compared to the reactions in the right ears of the vehicle-treatedmice (FIG. 7a). When the effect on leukocyte infiltration associatedwith the contact hypersensitivity reactions was assessed (FIG. 8a), theresults were similar. Reactions in the spiperone-treated right ears werediminished by 76% compared to the right ears of vehicle-treated mice,whereas reactions in the left ears of mice treated on the right earswith spiperone were reduced only 22% compared to those in the left earsof vehicle-treated mice.

A lower concentration of spiperone, applied topically to the right ear 2hours before (FIG. 7b and 8b) or 22 hours after (FIGS. 7c and 8c) haptenchallenge was also tested. The results demonstrate that the lowerconcentration of spiperone inhibited the majority of the tissue swellingand leukocyte infiltration associated with contact hypersensitivityreactions elicited at the site of treatment (the right ear), but had nosignificant effect on the intensity of the reactions elicited by thesame dose of hapten applied to the contralateral (left) ear. Note thattreatment with either vehicle had little or no effect on the responses(FIGS. 7 and 8).

Although topical application of spiperone was extremely effective indiminishing both the tissue swelling and the leukocyte infiltrationassociated with contact hypersensitivity reactions, these effects wereobserved in the absence of detectable alterations in the behavior of themice. In contrast to mice treated systemically with spiperone, the micetreated topically with this agent appeared active and retainedapparently normal interest in food and water.

To evaluate the effect of topical treatment with spiperone on contacthypersensitivity reactions elicited with a different hapten, the effectof topical treatment with a 0.5% suspension of spiperone on the contacthypersensitivity reactions elicited with DNFB was examined. Topicaltreatment with spiperone significantly diminished the tissue swellingassociated with reactions to DNFB (by 45%, FIG. 9) and had an even moresignificant effect on leukocyte infiltration (a reduction of 71%compared to right ears of vehicle-treated mice, FIG. 10). At this doseof spiperone and with this hapten, the effect of spiperone on reactionselicited in the left ears of mice treated on the right ears with thedrug were modest (28% reduction in tissue swelling and 18% reduction inleukocyte infiltration compared to values for the left ears ofvehicle-treated mice, FIG. 9 and 10). In fact, in this experiment, theeffect of spiperone applied to the right ears on the leukocyteinfiltration associated with reactions elicited in the left ears was notsignificant (p>0.05).

EXAMPLE 2 Comparison of Immunosuppressant versus Anti-inflammatoryactivity.

Mice were sensitized to oxazolone as described in Example 1. Three dayslater, slow release indomethacin pellets (0.05 mg, 3 week release) wereimplanted subcutaneously under light ether anesthesia. The dose ofindomethacin delivered by these pellets has been previously shown tocompletely block prostaglandin synthesis in mice, by Jun, D. D., et al.,J. Invest. Dermatol. 90:311 (1988).

Three days later, mice were challenged for contact hypersensitivity asin example 1. When the hypersensitivity response was assessed 24 hourslater, by measurements of tissue swelling and leukocyte infiltration,indomethacin was shown to have no significant effect on the response.These data show that a classic anti-inflammatory agent, indomethacin,cannot suppress the immunologically specific oxazolone induced contacthypersensitivity response.

Modifications and variations of the present invention relating tomethods for the treatment of pathology associated with immune responsesthat includes topical administration of an effective amount of spiperoneor a spiperone derivative will be obvious to those skilled in the artfrom the foregoing detailed description of the invention. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. A method for the treatment of a cutaneous, ocular, ormucosal pathology associated with an immune response in a human or othermammal comprising topically administering an effective amount of acompound selected from the group consisting of spiperone and a spiperonederivative of the formula: ##STR5## wherein R₁ =H, CH₃ --, C₆ H₅ --,cyclohexyl, 4--(OCH₃)C₆ H₄ --, 3--(CH₃)C₆ H₄ --, 4--(CH₃)C₆ H₄ --,4-X-C₆ H₄ --, (CH₃)₂ CH--, CH₃ (CH₂)₃ --, (CH₃)₂ CHCH₂ --, CH₃ CH₂CH(CH₃)--, (CH₃)₃ C--; or Y--CH₂ (CH₂)_(m) --.R₂ =H or CH₃ ; R₃ =H, CH₃,CH₃ CH₂ --, CH₃ CH₂ CH₂ --, (CH₃)₂ CH--, CN(CH₂)₂ --, or CH₃ (CH₂)₂ --;R₄ =H, C₆ H₅ CH(CH₂ CH₃)CH₂ --, C₆ H₅ CH(CH₃)(CH₂)₂ --, C₆ H₅ CH₂CH(CH₃)CH₂ --, C₆ H₅ CH₂ CH₂ CH(CH₃)--, C₆ H₅ CH(CH₃)(CH₂)₃ --, 4--CH₃C₆ H₄ CH(CH₃)(CH₂)₃ --, 4--(CH₃ O)C₆ H₄ CH(CH₃)(CH₂)₃,4--X--C₆ H₄CH(CH₃)CH₂ --, 4--X--C₆ H₄ CH(CH₂ CH₃)CH₂ --,4--X--C₆ H₄ CH(CH₃)(CH₂)₂--, 4--X--C₆ H₄ --CH(CH₃)(CH₂)₃ --,C₆ H₅ CH(OCH₃)(CH₂)₂ --, ##STR6## C₆H₅ CO(CH₂)₃ --,C₆ H₅ CO(CH₂)₄ --, 4--(CH₃)C₆ H₄ CO(CH₂)₃ --, 4--(CH₃O)C₆ H₄ CO(CH₂)₃ --, 4--X--C₆ H₄ CO(CH₂)₃ --, 4--X--C₆ H₄ CO(CH₂)₃ --,2--thienyl--CO(CH₂)₃ --, AR₁ --CH(CH₂)_(n) --, Ar, -- XC₆ H₄C(CH₃)CH(CH₂)₂ --, where the configuration about the double bond is cisor trans, 4-XC₆ H₄ C(CH₃)CHCH₂ --, where the configuration about thedouble bond is cis or trans, 4--X--C₆ H₄ COCH=CHCH₂ --, or Y--CH₂(CH₂)_(s--).wherein n=3 or 4; m is between 1 to 4; s is between 1 to 6;X=H, F, Cl, Br, I, OCH₃, SO₃, or NH₂ ; Y=H, F, Cl, Br, I, SO₃, PO₄ ⁼,OH, SH, SCH₃, CH₃ SO₂, NH₂ -CO₂ ; and each of Ar and Ar₁ is,independently, H, C₆ H₅ --, 4--(CH₃)C₆ H₄ --, 4-(CH₃ O)C₆ H₄ --,4--X--C₆ H₄ --, 3--(CH₃)C₆ H₄ --, 2-thienyl, or 4--X--C₆ H₄ CH₂ --; orits pharmaceutically acceptable salt, in a pharmaceutically-acceptablediluent or carrier for topical application.
 2. The method of claim 1wherein the mammal is a human.
 3. The method of claim 1 wherein thepathology is oral or mucosal and the carrier is a mouthwash.
 4. Themethod of claim 1, wherein the pathology is oral or mucosal, an the thespiperone or spiperone derivative is administered in a solution that isswished in the mouth and spit out.
 5. The method of claim 1 wherein thecompound in combination with an ophthalmic carrier is topically appliedto the eye.
 6. The method of claim 1, wherein the compound is appliedcutaneously.
 7. The method of claim 1, wherein the compound is appliedto mucosal membranes.
 8. The method of claim 1, wherein the daily doseof compound is between 0.1 milligrams and 120 grams.
 9. The method ofclaim 1, wherein the compound is applied in a concentration between0.001% and 50%.
 10. The method of claim 1 wherein the compound isadministered in a time release formulation.
 11. The method of claim 1,wherein the compound is administered via a retention enema.
 12. Themethod of claim 1, wherein the compound is administered in combinationwith another compound selected from the group consisting of antivirals,antifungals, antibiotics, anti-inflammatories, and otherimmunosuppressants and bronchodilators or other therapeutic agents forasthma.